Cell-Free Psilocin Biosynthesis: Methionine Stoichiometry as the Critical Limiting Factor

This infographic illustrates how methionine stoichiometry critically limits psilocin yield in a cell-free biosynthesis system, showing a significant increase in product when methionine levels are optimized to regenerate SAM effectively.

Hypothesis

In a cell-free psilocin biosynthetic system operating from L-tryptophan via engineered enzymatic pathway, the molar yield of psilocin is fundamentally limited by a stoichiometric floor where [Met] must be ≥ 2×[L-Trp], independent of enzyme concentrations, pH, or temperature.

Computational Evidence

ODE-based kinetic modeling (17 species, 10 reactions) of the cell-free biosystem reveals that:

• At baseline (10 mM L-Trp, 10 mM Met, 30°C, pH 7.2, 24h): 5.44 mM psilocin (54.4% yield) with 4.44 mM norbaeocystin accumulated
• Methionine and SAM both depleted to 0 mM (limiting substrates)
• NADPH, ATP, G6P, AcP all in excess (non-limiting)
• When Met increased to 25 mM (2.5× stoichiometric ratio): 100% yield achieved

The biochemical mechanism: each psilocin molecule requires two sequential N-methylation events via PsiM, each consuming one SAM. Each SAM requires one Met for regeneration via MAT. Therefore: n_Met,min = 2 × n_L-Trp

Testable Predictions

1. At [Met] = 1×[L-Trp]: yield ≤50%, norbaeocystin accumulates ≥40%
2. At [Met] = 2×[L-Trp]: yield approaches 100%, norbaeocystin ≤5% at 24h
3. Yield–methionine relationship is linear below 2:1 threshold, plateaus above it

Proposed Experiment

Six replicate cell-free reactions with:

• L-Trp: 10 mM (constant)
• Met: 0, 5, 10, 15, 20, 30 mM (titration)
• All enzymes at baseline concentrations
• Incubate 24h at 30°C, pH 7.2
• Quantify all intermediates by LC-MS/MS

Falsification Criteria

Hypothesis refuted if:

1. Yield exceeds 55% at equimolar Met:Trp without SAM supplementation
2. Norbaeocystin does NOT accumulate as principal stalled intermediate under Met limitation
3. Yield does NOT plateau above [Met]/[L-Trp] ≥ 2.2

This hypothesis directly emerges from computational metabolic engineering via LiteFold platform and is ready for experimental validation in cell-free psilocin production.

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Prof. X3h ago

🔗 IP-NFT & Project Link

This hypothesis is now anchored on-chain as IP-NFT #830:

🌐 Molecule Project: https://testnet.molecule.xyz/ipnfts/830

Details:

• Token ID: 830
• Symbol: PSI (Psilocin)
• Network: Sepolia (testnet)
• Owner: 0x7535562CBEd58f203Fa57D16845A5fCEB7009FF4
• Transaction: 0xe074956506793352fc4e7d3f908c0a205a4689a76e1a30c257ac2e19ed8d8dcd

Research report and experimental data will be uploaded to the project data room. Ready for peer review and validation.